Iron-sulfur [Fe-S] proteins are found in all classes of biological organisms, from the most primitive to the most advanced. Their predominant role is electron transport. The broad long- term goal of this research is to understand the structures, properties and functions of [Fe-S] clusters in [Fe-S] clusters in [Fe-S] proteins. We propose to pursue this goal via studies of site-directed mutants of Azotobacter vinelandii ferredoxin I (AvFdI). Native AvFdI contains 106 amino acids and two [Fe-S] clusters: one Fe4S4Cys4 cluster and one F33S4Cys3 cluster. Its structure is known from X-ray crystallography. We propose to study mutants of AvFdI designed to elucidate the following issues; (i) how does the polypeptide of an [Fe-S] protein control the redox potential of its [Fe-S] cluster(s)? (ii) what are the structures and properties of heterogeneously-ligated [Fe-S] clusters, in which ligands other than cysteine are present? (iii) what is the relationship between the primary sequence of an [Fe-S] protein and the structure(s) of the [Fe-S] cluster(s) it supports? (iv) what are the chemical reactions of [Fe-S] clusters in [Fe-S] proteins in addition to reversible oxidation-reduction and what is their biological relevance? Mutants will be characterized using X-ray crystallography, electrochemistry and a variety of spectroscopic methods (including EPR, NMR, MCD and Mossbauer spectroscopies). A major focus of the proposed research is the evaluation of a new theoretical methodology for the prediction of the redox potentials of [Fe- S] proteins. Initial studies have shown that quantitatively realistic calculations of the tuning of [Fe-S] cluster redox potentials by their protein environments are now practicable, permitting for the first time a detailed understanding of the structure-function relationship in electron- transport [Fe-S] proteins.